The present invention relates to the discovery of a novel IL-7/HGFxcex2 hybrid cytokine complex referred herein as a pre-pro-B Cell Growth Stimulating Factor, or PPBSF, and methods for its production from recombinant or naturally-derived IL-7 and HGFxcex2.
Hepatocyte growth factor (HGF), also called scatter factor SF, is a heparin-binding glycoprotein that is secreted as a biologically inert single chain (pro-HGF) and is converted to its bioactive form by targeted protease digestion to an active disulfide-linked heterodimer. HGF is a natural ligand for the c-MET proto-oncogene product of a novel family of heterodimeric receptor tyrosine kinases that include Ron, Sea and Sex. It is a pleiotropic factor derived from the mesenchyme that regulates epithelial, neural, endothelial, muscle and hemopoietic cell growth, motility, morphogenesis and regeneration in many tissues and organs. The importance of HGF is seen in transgenic mice homozygous for a null mutation in the HGF gene. Such mice do not survive beyond fifteen days of embryonic development.
Mature bioactive HFG is a heterodimer consisting of a 60 kD alpha and 30 kD beta chain held together by a single disulfide bond. Structure function analysis indicates that the beta chain of HGF is required for mitogenic activity, whereas the receptor-binding domain is located in the alpha chain. Its primary structure is highly conserved among mouse, rat, human and other species. The alpha chain contains a hairpin loop at its amino terminus and four unique domains known as xe2x80x9ckringlesxe2x80x9d, while its beta chain contains a serine protease-like structure. Hence, HGF is closely homologous to plasminogen, but has no known protease activity due to mutation of the catalytic site.
HGF has been reported to be sequestered in the extracellular matrix (ECM) in vitro as well as in vivo, where it is bound to cell surface heparin sulfate glycosoaminoglycans. In general, HGF mRNA is expressed in stromal cells, whereas HGF receptor expression is mainly detected in epithelial and other parenchymal cells. This pattern suggests that HGF is an important paracrine mediator of the interaction between the parenchymal and stromal components of various tissues both during fetal development and in the maintenance of homeostasis in adult tissues.
Although a great deal is known about the actions of HGF in nonhemopoietic tissues, the role of HGF in the regulation of hematopoiesis, particularly lymphopoiesis, is fragmentary. HGF has been proposed to regulate hematopoiesis in mouse fetal liver and adult bone marrow in vivo, where it apparently can substitute for the stem cell factor (SCF) and c-kit system. HGF is produced by bone marrow (BM) stromal cells and synergizes with IL-3 or GMCSF to support the growth of hemopoietic progenitor cells (HPCs) and myeloid tumor cell lines, all of which express the HGF receptor, c-MET. In the presence of erythropoietin, HGF induces the formation of colonies along the erythroid lineage, whereas in the presence of erythropoietin plus SCF, HGF supports the growth of multipotent colonies. Similarly, upregulation of the HGF receptor on primitive hematopoietic cells may be induced by IL-11; and the synergistic interaction of these two cytokines may result in in vitro colony formation by hemopoietic stem cells (HSCs). However, HGF alone does not appear to stimulate proliferation of hemopoietic precursors. The latter may be attributed to enhancement by HGF of signal transduction by lineage-specific cytokines.
HGF has been found to promote adhesion of HPCs to fibrinectin in vitro, and may be involved in a novel paracrine signaling pathway regulating integrin-mediated adhesion and migration of B cells in germinal centers. Messenger-RNA for c-MET has been identified in thymocytes as well as in early B-lineage cells in bone marrow. It is hypothesized that HGF may be involved in a novel paracrine signaling pathway that regulates integrin-mediated adhesion and migration of B-cells in germinal centers. Thus, HGF may be one of the long sought mediators of paracrine interactions between stromal and lymphohematopoietic cells. Furthermore, HGF seems to preferentially affect hematopoietic cells in a window of differentiation between multipotent progenitors and committed precursors. For example, the addition of HGF to fetal thymus organ cultures is known to increase the generation of mature T cells.
Interleukins are a class of proteins that induce growth and differentiation of lymphocytes and hematopoetic stem cells. One interleukin in particular, IL-7, has been demonstrated over the past decade to have an essential role in the development and differentiation of murine pre-B cells.
The nature of IL-7 involvement (if any) at earlier stages of B cell development remains controversial. While it has been proposed that IL-7 is capable of acting on primitive B220xe2x88x92 B cell progenitors in the presence of stem cell factor (SCF), most investigators have concluded that the principle B-lineage targets for IL-7 are pro-B cells and pre-B cells. The pre-B cells that do appear in IL-7 KO mice are abnormal as evidenced by their failure to up-regulate or express IL-7Rxcex1, TdT and Cxcexc. However, some redundancy may exist between the activities of IL-3, TSLP, and IL-7. Additionally, it has been suggested that the short-term maintenance of pre-pro-B cells, but not pro-B cells, depends on contact-mediated signals from BM stromal cells. Thus, in vivo treatment of mice with anti-IL-7 antibodies eliminates B-lineage subsets as early as the pro-B, but not the pre-pro-B, cell stage; a similar maturational arrest has been observed in mice having disrupted IL-7 receptor xcex1-chain genes (IL-7Rxcex1xe2x88x92/xe2x88x92); and the Tyr449 to Phe xcex1-chain point mutation suggests that the IL-7R transmits distinct signals for cell proliferation and IgH gene rearrangement. In contrast, von Freeden-Jeffry et al. (D. Exp. Med. 181: 1519 (1995)) found that both pre-pro-B cells and pro-B cells are well represented in BM of IL-7 gene-deleted mice; and Pribyl and LeBien (Proc. Nat. Acad. Sci. USA 93: 10348 (1996)) have reported that human B-lineage cells can be generated from fetal precursors in an IL-7-independent manner.
It must be cautioned that the presence of pre-pro-B cells in IL-7Rxcex1 chain (xe2x88x92/xe2x88x92) mice does not necessarily preclude the involvement of IL-7 at this developmental stage in normal animals. An alternative explanation is that the immediate precursors of pre-pro-B cells do not require an IL-7R-mediated signal to generate pre-pro-B cells. It must also be cautioned that the presence of pro-B cells in IL-7 gene-deleted mice does not exclude a physiological role for IL-7 in early B-lineage development; neither does it preclude the possibility that cytokines other than IL-7 use the IL-7R to stimulate proliferation and differentiation of early B-lineage precursors. Indeed, our recent studies in IL-7 KO mice (see Progress Report) have demonstrated that IL-7 is essential for upregulation of TdT and IL-7R xcex1-chain expression among early pro-B cells and for initiation of cxcexc expression in late pro-B cells. Therefore, while pro-B cell development occurs in IL-7 KO mice, such development is abnormal. Similar explanations may apply to conflicting reports regarding the need for IL-7 in normal human B cell ontogeny, although important species-specific differences may exist.
In prior studies, the present inventors have demonstrated that serum-free BM stromal cell conditioned medium (CM), as described in Nakumra et al., Nature 342: 440-443 (1989), Rubin et al., Biophysica Acta 1155: 357-371 (1993) and Zarnegar et al., J. Cell Biol. 129: 1177-1180 (1995), selectively stimulates the proliferation of early (TdTxe2x88x92) and late (TdT+) pre-pro-B cells from freshly-harvested rat BM and supports the generation (but not the proliferation) of pro-B cells. Furthermore, adsorption of CM with anti-IL-7 mAb removes this activity, whereas rIL-7 restores this activity to medium conditioned by BM stromal cells from IL-7 gene-deleted mice (xe2x88x92/xe2x88x92CM). Nonetheless, anti-IL-7 mAb is unable to neutralize the pre-pro-B cell growth-stimulating activity in IL-7 (+/+) CM or in rIL-7-supplemented (xe2x88x92/xe2x88x92) CM; and rIL-7, is unable to restore PPBSF activity to IL-7 (+/+) CM that has been adsorbed with anti-IL-7 mAb. The reason for these finding are not explained by the prior art discussed above.
The present inventors have discovered that the unique lymphopoietic properties of their BM lymphoid culture system was due to the presence of a self-assembling molecular complex of IL-7 and a second stromal cell-derived factor, a molecular complex previously unrecognized in the art. Biological properties of the self-assembling molecular complex, designated pre-pro-B cell growth stimulating factor, or PPBSF for short, suggest widespread medical applications.
Western blot analysis under reducing and nonreducing conditions directly demonstrates that PPBSF is a covalently-bound, Mr 55,000, heterodimer. The heterodimer comprises a non-IL-7 co-factor (coF) of about Mr 30,000 (determined using monoclonal antibodies derived from PPBSF-immunized IL-7 KO mice). PPBSF-coF has been found by the present inventors to be constitutively produced by BM stromal cells from IL-7 KO mice cultured under pro-B cell but not pre-B cell (i.e. Whitlock/Witte-type culture conditions). PPBSF has been found by the present inventors to xe2x80x9cprimexe2x80x9d pre-pro-B cells to proliferate in response to monomeric IL-7 in an anchorage-independent fashion by upregulating the expression of the IL-7Rxcex1 chain.
By both amino acid sequence analysis and reciprocal Western immunoblotting, it has now been discovered that the PPBSF-coF of PPBSF is the B-chain of HGF. In confirmation of the same, the bioactivity of native PPBSF has been found to be neutralized by antibodies to the HGFxcex2-chain. Although cDNA for pro-HGF had previously been cloned prior to the present invention, the xcex2-chain cDNA had not been isolated and cloned into appropriate expression vectors.
PCR amplification of the coding sequence of HGF in stromal cells from IL-7 KO mice resulted in the application of two transcripts of 2230 and 840 bp. The smaller product showing complete homology with the published mouse HGFxcex2 gene was subcloned into the mammalian expression vector pcDNA3.1 (+) and transfected into Chinese hamster ovary (CHO) cells. The HGFxcex2 gene was also subcloned into the prokaryotic fusion protein expression vector pCAL-n and transformed into E. coli BL21 (DE3).
The rHGFxcex2 DNA was purified by calmodulin affinity resin. Unexpectedly, rIL-7 spontaneously complexed with rHGFxcex2 in the presence of low molecular weight heparin sulfate (HS)-derived oligosaccharides (below about 3000 kD) to form a heterodimer having the functional activity of native PPBSF. However, because several naturally occurring variant HGFxcex2 produced by alternative splicing of the HGF gene have been identified, the precise form of HGFxcex2 represented in PPBSF remains to be determined, as does its origins (i.e. alternative splicing or duplication of the HGF gene), synthesis, assembly with IL-7; and display.
This is the first demonstration of a naturally occurring, or an artificially constructed, hybrid cytokine (i.e. a biomolecular or unimolecular complex of the bioactive portions of two or more disparate cytokines or growth factors). It also is the first demonstration of a bioactive form of IL-7 and HGFxcex2 that selectively supports the proliferation and subsequent differentiation of pre-pro-B cells. Although IL-7 plays an essential role in the development of early B lymphocytes, IL-7 alone doesn""t support the proliferation of pre-pro-B cells. Although HGF can synergize with IL-3, GM-CSF or erythropoietin to support the growth of HPCs, myeloid cell lines, and erythroid cells, respectively, it has not been reported to play a direct role in the early B-cell development. Hence, the discovery of the IL-7/HGFxcex2 complex not only provides a reagent that regulates the earliest stages of B-lymphocyte development in bone marrow, but it may presage the existence of a series of other naturally occurring hybrid cytokines as well as the artificial creation of hybrid cytokines with unique pharmacological properties. In addition, the existence/creation of hybrid cytokines may render pleiotropic growth factors lineage-specific, thereby directing the commitment of hemopoietic and other pluripotent stem cells to development along selective pathways.
xe2x80x9cIL-7/HGFxcex2 complex,xe2x80x9d as used herein, refers both to a bimolecular protein complex which features both the IL-7 (Interleukin-7) and HGFxcex2 polypeptides, biologically-active variants thereof, and to a unimolecular protein which includes the bioactive portions of IL-7 and HGFxcex2 connected with a flexible linker. The expression xe2x80x9clinkerxe2x80x9d relates to linkers of any kind, which are suitable for the binding of polypeptides.
Examples of such linkers include but are not limited to a disulfide-bridge connecting amino acids from both polypeptides; heparin or heparan sulfate-derived oligosaccharides (glycosoaminoglycans) connecting both polypeptides; bifunctional or chemical cross-linkers; and a peptide or polypeptide linker. The unimolecular protein can also be a fusion polypeptide. For example, a polypeptide featuring the bioactive portions of IL-7 and HGFxcex2 can be fused with each other, and the linker can be a disulfide-bridge produced by the two polypeptides.
PPBSF has been found to selectively stimulate the proliferation of pre-pro-B cells and to support the generation of pro-B cells (the next recognized stage in early B-lymphocyte development). PPBSF xe2x80x9cprimesxe2x80x9d pre-pro-B cells to proliferate in response to monomeric IL-7 in an anchorage-independent fashion by upregulating the expression of the IL-7 receptor (R) xcex1 chain. PPBSF also upregulates the expression of terminal deoxynucleotidyl transferase (TdT) and initiates the expression of cytoplasmic immunoglobulin mxcexc heavy chain (cxcexc). PPBSF also stimulates the proliferation of thymocytes.
The IL-7/HGFxcex2 complex can be isolated from natural sources, e.g., mammalian tissues or cell lines which are known to be a source of cytokines or growth factors. It may also be formed from recombinant and/or natural components as shown herein. PPBSF was shown to be expressed by bone marrow stromal cells in our pro-B cell culture system. Alternatively, PPBSF can be reconstituted from products of prokaryotic or eukaryotic expression of exogenous DNA sequences i.e., derived by recombinant means.
The present invention also includes biologically-active variants of the IL-7 or HGFxcex2 complex. Such variants may include any homologous peptide to either IL-7 or HGFxcex2, for example including substitution analogs wherein one or more amino acids have been substituted with different amino acids, deletion analogs wherein one or more amino acids have been deleted, and addition analogs wherein one or more amino acids have been added. Deletions and additions of one or more amino acids are made either within an internal region of the polypeptide or at the amino or carboxyl terminal ends. Additional potential variations include other heterodimeric (or multimeric) cytokine complexes containing IL-7 and/or HGF (xcex1 and/or xcex2 chains), and other hybrid cytokines unrelated to either IL-7 or HGF, whether naturally occurring or artificially created, including those that bind to the receptors for HGF, IL-7, and/or xcex3c.
Western immunoblotting showed that PPBSF was a covalently-linked heterodimer of IL-7 and an Mr 30,000 cofactor. Partial NH2-terminal amino acid sequence analysis of purified PPBSF cofactor showed the first 15 of 17 amino acid residues were identical to the published sequence of mouse HGF xcex2 chain. Western blot analysis confirmed the identity of PPBSF cofactor as the xcex2 chain of HGF.
In conjunction with the invention, the present inventors have: (1) established a pro-B cell culture system that selectively generates large number of pre-pro-B cells and pro-B cells from rat, mouse and human bone marrow; (2) demonstrated that medium conditioned by BM stromal cells in our pro-B cell culture system selectively supports the development of pre-pro-B cells and pro-B cells in vitro; (3) demonstrated the existence in conditioned medium of a non-IL-7 component of PPBSF by anti-IL-7 antibody neutralization and adsorption experiments; (4) demonstrated that PPBSF is a covalently-linked heterodimer of IL-7 and a Mr. 30,000 cofactor by Western immunoblot analysis under reducing and non-reducing conditions; (5) demonstrated that PPBSF is a self-aggregating complex of IL-7 and a Mr. 30,000 cofactor by addition of IL-7 to conditioned medium from IL-7 gene-deleted mice; (6) demonstrated that PPBSF, but not IL-7 or PPBSF cofactor alone, upregulates the expression of IL-7Rxcex1, TdT and cxcexc on/in pro-B cells from IL-7 gene-deleted mice and xe2x80x9cprimesxe2x80x9d then to proliferate in response to monomeric IL-7; (7) developed neutralizing monoclonal antibodies specific for the PPBSF cofactor; (8) identified the PPBSF cofactor as the xcex2 chain of HGF/SF by amino acid analyses and reciprocal Western blotting; (9) cloned the HGFxcex2 cDNA into mammalian and prokaryotic xpression vectors and expressed the protein in mammalian (CHO) and prokaryotic e. coli BL21 (DE3) cells; and (10) demonstrated that rIL-7 spontaneously complexes with r HGFxcex2 in the presence of low molecular weight heparin sulfate (HS)-derived oligosaccharides to form a heterodimer having the functional activity of native PPBSF.